Nucleotide monomers containing 8-azapurin bases or a derivative thereof, their preparation and their use in making modified olignonucleotides

ABSTRACT

The invention relates to novel modified oligonucleotides which contain at least one 8-azapurine base and form more stable hybridization complexes with nucleic acids; To a process for their preparation, and to their use as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology, and as a pharmaceutical or diagnostic agent.

This application is a continuation of application Ser. No. 08/431,777, filed May 1, 1995, now abandoned.

The present invention relates to novel oligonucleotides which contain modified bases and which possess valuable physical, biological and pharmacological properties, to a process for their preparation, and to their use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for detecting nucleic acids, as aids in molecular biology, and as a pharmaceutical or a diagnostic agent.

Numerous chemical modifications of oligonucleotides are known from the literature. These modifications can affect the sugar-phosphate skeleton or the nucleotide bases. Reviews of the state of the art are provided, for example, by Uhlmann & Peyman, Chem. Rev. 1990, 90, 543 and Milligan et al., J. Med. Chem. 1993, 36, 1923.

As a rule, it is necessary to modify the oligonucleotides chemically, since unmodified oligonucleotides are very rapidly degraded by nucleolytic activities both in cells and in cell culture medium. Stabilization against nucleolytic degradation can be achieved by replacing the sugar-phosphate backbone, or by modifying the phosphate bridge, the sugar component or the nucleotide base Milligan et al., see above, and Uhlmann & Peyman, see above!.

In addition to modifications which lead to oligonucleotides which have increased stability towards nucleolytic degradation, modifications are also of interest which alter the hybridization behavior of the modified oligonucleotides such that these oligonucleotides are able, for example, to form more stable hybridization complexes (duplexes) with intracellular nucleic acid (so-called target nucleic acids). It is possible to alter the hybridization properties of oligonucleotides by, for example, modifying their bases. The altered hybridization properties of such modified oligonucleotides can be recognized, for example, by the fact that the melting temperature (T_(m) value) of the duplexes is altered as compared with when unmodified oligonucleotides are used.

Thus, PCT Application WO 92/002258 describes pyrimidine-modified oligonucleotides which, however, exhibit a decreased rather than an increased binding affinity for single-stranded and double-stranded target nucleic acids. However, PCT Application WO 93/10820 also discloses oligonucleotides which contain modified uracil bases and cytosine bases and which can form duplex or triplex structures with the target nucleic acids which are more stable than those achieved using non-modified oligonucleotides. The hybridization properties of synthetic dodecamer oligonucleotides which contain the base analog pyridopyrimidine have also been investigated Inoue et al. (1985), Nucleic Acid Res., 13, 7119-7129!. Oligonucleotides which contain the base analog 2-aminoadenine have also been reported to have improved hybridization properties Chollet et al., (1988), Nucleic Acid Research, 16, 305-317!.

The object of the present invention is, therefore, to make available novel oligonucleotides which possess advantageous properties.

It has now been found, surprisingly, that oligonucleotides which contain at least one 8-aza-purine base, for example 8-azaguanine or 8-azaadenine, form hybridization complexes with the target nucleic acids which are markedly more stable than those formed by comparable oligonucleotides which contain the unmodified purine bases.

The invention thus relates to oligonucleotides of the formula I ##STR1## and the physiologically tolerated salts thereof, in which R¹ is hydrogen, C₁ -C₁₈ -alkyl, C₂ -C₁₈ -alkenyl, C₂ -C₁₈ -alkynyl, C₂ -C₁₈ -alkylcarbonyl, C₃ -C₁₉ -alkenylcarbonyl, C₃ -C₁₉ -alkynylcarbonyl, (C₆ -C₁₄) -aryl-(C₁ -C₈) alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIa ##STR2## R^(1a) is hydrogen, C₁ -C₁₈ -alkyl, C₂ -C₁₈ -alkenyl, C₂ -C₁₈ -alkynyl, C₂ -C₁₈ -alkylcarbonyl, C₃ -C₁₉ -alkenylcarbonyl, C₃ -C₁₉ -alkynylcarbonyl, (C₆ -C₁₄) -aryl-(C₁ -C₈)-alkyl, or a radical of the formula IIb ##STR3## R² is hydrogen, hydroxyl, C₁ -C₁₈ -alkoxy, C₁ -C₆ -alkenyloxy, in particular allyloxy, halogen, azido or NH₂ ;

a is oxy or methylene;

n is an integer from 3 to 99;

W is oxo, thioxo or selenoxo;

V is oxy, sulfanediyl or imino;

Y is oxy, sulfanediyl, imino or methylene;

Y' is oxy, sulfanediyl, imino, (CH₂)_(m) or V(CH₂)_(m), in which

m is an integer from 1 to 18;

X is hydroxyl or mercapto;

U is hydroxyl, mercapto, SeH, C₁ -C₁₈ -alkoxy, C₁ -C₁₈ alkyl, C₆ -C₂₀ -aryl, (C₆ -C₁₄) -aryl-(C₁ -C₈) -alkyl, NHR³, NR³ R⁴ or a radical of the formula III

    (OCH.sub.2 CH.sub.2).sub.p O(CH.sub.2).sub.q CH.sub.2 R.sup.5(III)

in which

R³ is C₁ -C₁₈ -alkyl, C₆ -C₂₀ -aryl, (C₆ -C₁₄)-aryl-(C₁ -C₈)alkyl, or 2-(CH₂)_(c) -- NH(CH₂)_(c) !_(d) --NR⁶ R⁶, in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and R⁶ are, independently of each other, hydrogen or C₁ -C₆ -alkyl or C₁ -C₄ -alkoxy-C₁ -C₆ -alkyl;

R⁴ is C₁ -C₁₈ -alkyl, C₆ -C₂₀ -aryl or (C₆ -C₁₀)-aryl-(C₁ -C₈)-alkyl, or, in the case of NR³ R⁴, is, together with R³ and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the group O, S and N,

p is an integer from 1 to 100,

q is an integer from 0 to 22,

R⁵ is hydrogen or a functional group such as, for example, hydroxyl, amino, C₁ -C₁₈ -alkylamino, COOH, CONH₂, COO(C₁ -C₄)-alkyl or halogen;

Z and Z' are, independently of each other, hydroxyl, mercapto, SeH, C₁ -C₂₂ -alkoxy, --O--(CH₂)_(b) --NR⁶ R⁷, in which b is an integer from 1 to 6, and R⁷ is C₁ -C₆ -alkyl, or R⁶ and R⁷, together with the nitrogen atom carrying them, form a 3-6-membered ring, C₁ -C₁₈ -alkyl, C₆ -C₂₀ -aryl, (C₆ -C₁₄)-aryl-(C₁ -C₈)-alkyl, (C₆ -C₁₄)-aryl-(C₁ -C₈)-alkoxy, where aryl is also heteroaryl, and aryl is optionally substituted by 1, 2 or 3 identical or different radicals from the group carboxyl, amino, nitro, C₁ -C₄ -alkylamino, C₁ -C₆ -alkoxy, hydroxyl, halogen and cyano, C₁ -C₁₈ -alkylmercapto, NHR³, NR³ R⁴, a radical of the formula III or a group which favors intracellular uptake or which serves as the label of a DNA probe, or, when the oligonucleotide analog hybridizes to the target nucleic acid, attacks the latter with binding, crosslinking or cleavage, and the curved bracket indicates that R² and the adjacent phosphoryl radical can be located either in the 2' and 3' positions or else, conversely, in the 3' and 2' positions, where each nucleotide can be present in its D or L configuration and the base B can be located in the α or β position, where B are, independently of each other, a base which is customary in nucleotide chemistry, for example natural bases such as adenine, cytosine, thymine, guanine, uracil or hypoxanthine, or unnatural bases such as, for example, purine, 8-azapurine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N⁴ N⁴ -ethanecytosine, N⁶ N⁶ -ethano-2,6-diaminopurine, pseudoisocytosine, 5-methylcytosine, 5-fluorouracil, 5-(C₃ -C₆)-alkynyluracil, 5-(C₃ -C₆)-alkynylcytosine, or their prodrug forms, where at least one B is a base of the formula IV ##STR4## in which E and F are, independently of each other, H, OH or NH₂.

Compounds of the formula I are preferred, in which E is NH₂ and F is OH, or E is H and F is NH₂.

Compounds of the formula I are particularly preferred in which E is NH₂ and F is OH.

Compounds of the formula I are also preferred in which the base is located in the β position on the sugar, the nucleosides are present in the D configuration, R² is located in the 2' position and a is oxy.

When attached to complementary nucleic acids (target nucleic acids), the novel oligonucleotides exhibit a binding affinity which is improved as compared with that exhibited by the natural oligonucleotides. If these novel oligonucleotides are to be used therapeutically, it is advantageous if additional modifications, for example of the phosphate backbone, the ribose unit or the oligonucleotide ends, are introduced into them J. S. Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press, E. Uhlmann et al., see above!. For example, the novel oligonucleotides are protected even more effectively against nuclease attack, which is advantageous, if modifications, which are known per se, are made to their sugar-phosphate backbone.

Compounds of the formula I are also preferred, therefore, in which V, Y, Y' and W have the meaning of thioxo, selenoxo, oxy, oxo, sulfanediyl, imino or methylene, and U has the meaning of hydroxyl, mercapto or methyl. The latter compounds are very particularly preferred if R² is also hydroxyl or hydrogen, in particular hydrogen.

Compounds of the formula I in which R¹ and R^(1a) are hydrogen also represent a preferred embodiment.

Compounds of the formula I are very particularly preferred in which R¹ and/or R^(1a) is/are hydrogen, R² is hydroxyl or hydrogen, U is hydroxyl or mercapto and V, Y, Y' and W have the meaning of thioxo, oxy, oxo or hydroxyl.

Protective groups which are customary in nucleotide chemistry are understood to mean, for example, amino protective groups, hydroxyl protective groups or other protective groups, as described in E. Sonveaux, 1986, Bioorganic Chemistry, 14, 274-325 or S. L. Beaucage et al., 1992, Tetrahedron, 48, 2223-2311!.

Alkyl, alkenyl or alkynyl can be straight-chain or branched.

Cycloalkyl is also understood to mean alkyl-substituted rings.

Examples of (C₆ -C₂₀)-aryl are phenyl, naphthyl or biphenyl, preferably phenyl.

Heteroaryl is understood to mean, in particular, radicals which are derived from phenyl or naphthyl and in which one or more CH groups are replaced by N, and/or in which at least two adjacent CH groups are replaced by S, NH or O (with the formation of a five-membered aromatic ring). In addition, one or both atoms of the condensation site of bicyclic radicals can be N atoms (as in indolizinyl).

Furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, indazolyl, quinolyl, isoquinolyl, phthalazinyl, quinoxalinyl, quinazolinyl and cinnolinyl are, in particular, regarded as heteroaryl.

Physiologically tolerated salts of compounds of the formula (I) are understood to be both inorganic and organic salts, as described in Remington's Pharmaceutical Sciences (17th edition, page 1418 (1985)).

For reasons of physical and chemical stability, and solubility, sodium salts, potassium salts, calcium salts and ammonium salts, inter alia, are preferred for acid groups.

The invention is not limited to α- and β-D- and L-ribofuranosides, α- and β-D- and L-deoxyribofuranisides and corresponding carbocyclic five-membered ring analogs, and, instead, also applies to oligonucleotide analogs which are constructed from other sugar building blocks, for example xylofuranose and arabinofuranose derivatives, ring-expanded and ring-contracted sugars, acyclic or ring-bridged sugar derivatives, or suitable sugar derivatives of another type. Furthermore, the invention is not limited to the derivatives of the phosphate radical which are given by way of example in formula I, and, instead, also relate to the known dephospho derivatives.

The novel oligonucleotides can, consequently, be derived by modifying the natural structure in a wide variety of ways. Examples of these modifications, which can be introduced by methods which are known per se, are:

a) Modifications of the phosphate bridge

The following may be mentioned by way of example: phosphorothioates, phosphorodithioates, methyl phosphonates, phosphoramidates, boranophosphates, methyl phosphates, ethyl phosphates and phenyl phosphonates. Phosphorothioates, phosphorodithioates and methyl phosphonates are preferred modifications of the phosphate bridge.

b) Replacement of the phosphate bridge

The following may be mentioned by way of example: replacement with formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylenesulfone or silyl groups. Replacement with formacetals and 3'-thioformacetals is preferred.

c) Modifications of the sugar

The following may be mentioned by way of example: α-anomeric sugars, 2'-O-methylribose, 2'-O-butylribose, 2'-O-butylribose, 2'-O-allylribose, 2'-fluoro-2'-deoxyribose, 2'-amino-2'-deoxyribose and α-arabinofurose, and carbocyclic sugar analogs. The preferred modification is that with 2'-O-methylribose and 2'-O-n-butylribose.

d) Modifications of the sugar and of the phosphate bridge

The peptide nucleic acids (PNAs), in which the sugar/phosphate backbone is replaced by an aminoethylglycine backbone, and also the carbamate-bridged morpholino oligomers, may be mentioned by way of example.

e) Other modifications of the bases, in particular of the pyrimidine bases

The following may be mentioned by way of example: 5-propynyl-2'-deoxyuridine, 5-propynyl-2'-deoxycytidine, 5-hexynyl-2'-deoxyuridine, 5-hexynyl-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine, 5-fluoro-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxyuridine, 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine. 5-Propynyl-2'-deoxyuridin, 5-hexynyl-2'-deoxyuridine, 5-hexynyl-2'-deoxycytidine and 5-propynyl-2'-deoxycytidine are preferred modifications.

f) 3'-3' inversions and 5'-5' inversions e.g. M. Koga et al., J. Org. Chem. 56 (1991) 3757!

g) 5'conjugates and 3' conjugates.

Examples of groups which favor intracellular uptake are different lipophilic radicals such as --O--(CH₂)_(x) --CH₃, in which x is an integer from 6 to 18, --O--(CH₂)_(n) --CH═CH--(CH₂)_(m) --CH₃, in which n and m, independently of each other, are an integer from 6 to 12, --O--(CH₂ CH₂ O)₄ --(CH₂)₉ --CH₃, --O--(C₂ CH₂ O)₈ --(CH₂)₁₃ --CH₃ and --O--(CH₂ CH₂ O)₇ --(CH₂)₁₅ --CH₃, and also steroid residues such as cholesteryl, or vitamin residues such as vitamin E, vitamin A or vitamin D, and other conjugates which exploit natural carrier systems, such as cholic acid, folic acid, 2-(N-alkyl, N-alkoxy)-aminoanthraquinone and conjugates of mannose, and peptides of the corresponding receptors which lead to receptor-mediated endocytosis of the oligonucleotides, such as EGF (epidermal growth factor), bradykinin and PDGF (platelet-derived growth factor). Labeling groups are understood to mean fluorescent groups, for example of dansyl(=N-dimethyl-1-aminonaphthyl-5-sulfonyl) derivatives, fluorescein derivatives or coumarin derivatives, or chemiluminescent groups of, for example, acridine derivatives, and also the digoxygenin system, which can be detected by way of ELISA, the biotin group which can be detected by way of the biotin/avidin system, or else linker arms having functional groups which permit subsequent derivatization with detectable reporter groups, for example an aminoalkyl linker which is reacted with an active acridinium ester to form a chemiluminescent probe. The following are typical labeling groups: ##STR5##

Oligonucleotide analogs which bind to or intercalate with nucleic acids and/or cleave or crosslink them contain, for example, conjugates of acridine, psoralene, phenanthridine, naphthoquinone, daunomycin or chloroethylaminoaryl. Typical intercalating and crosslinking residues are: ##STR6##

Examples of NR³ R⁴ groups, in which R³ and R⁴, together with the nitrogen atom carrying them, form a 5- or 6membered heterocyclic ring which additionally contains a further heteroatom, which may be mentioned are the morpholinyl residue and the imidazolidinyl residue.

The invention furthermore relates to the compound of the formula V ##STR7## in which V is oxy, sulfanediyl or imino;

Y^(b) is oxy, sulfanediyl, imino or methylene;

a is oxy or methylene;

R^(2b) is hydrogen, OR¹², C₁ -C₁₈ -alkoxy, C₁ -C₆ -alkenyloxy, in particular allyloxy, halogen, azido or NR¹⁰ R¹¹ ;

R¹ is a protective group which is customary in nucleotide chemistry;

R^(1b) is a radical of the formula IIc or IId ##STR8## in which U is O--R⁷ or S--R⁷ ;

Q is a radical --NR⁸ R⁹,

R⁷ is --(CH₂)₂ --CN;

R⁸ and R⁹ are identical or different and are C₁ -C₆ -alkyl, in particular isopropyl or ethyl, or, together with the nitrogen atom carrying them, are a 5-9-membered heterocyclic ring which can additionally contain a further heteroatom from the group O, S and N, in particular ##STR9## E' and F' are, independently of each other, H, OH or NR¹⁰ R¹¹, R¹⁰ and R¹¹ are identical or different and are hydrogen or an amino protective group which is customary in nucleotide chemistry, or R¹⁰ and R¹¹ together form an amino protective group which is customary in nucleotide chemistry,

R¹² is a hydroxyl protective group which is customary in nucleotide chemistry, such as, for example, t-butyldimethylsilyl, triisopropylsilyl, o-nitrobenzyl, p-nitrobenzyl, tris(1-methylethyl)silyl or 2-fluorophenyl-4-methoxypiperidin-4-yl (FPMP),

and the curved bracket indicates that R² and the adjacent phosphoryl radical can be in the 2' and 3' positions or else, conversely, in the 3' and 2' positions.

Compounds of the formula (V) in which V, Y^(b) and a are oxy, R^(2b) is hydrogen or OR¹², in particular hydrogen, and R^(1b) is a radical of the formula (IIc) or (IId), where U is O--(CH₂)₂ --CN and R⁸ and R⁹ are identical or different and are isopropyl or ethyl, represent a preferred embodiment.

These compounds are very particularly preferred if, in addition, the base on the sugar is in the β position and R^(2b) is in the 2' position.

The invention also relates to compounds of the formula V in which linkage to the sugar residue takes place by way of the N2 atom of the 8-azapurine base.

Examples of preferred amino protective groups are acyl protective groups or amidine protective groups.

The invention also relates to compounds of the formula VI ##STR10## in which, independently of each other, U'=U"=U"' is hydroxyl or mercapto;

e and f are 0 or 1;

R¹³ is hydrogen or OH, and

E and F H, OH or NH₂,

where compounds of the formula VI are excepted in which U', U", U"', R¹³ and F are OH, E is NH₂ and e and f are 1.

Compounds of the formula VI are preferred in which U' is hydroxyl or mercapto, U"=U"' is hydroxyl, and e and/or f is/are 1.

In additiion, compounds of the formula VI are preferred in which

E is H, and F is NH₂, or, if E is NH₂, and F is OH, R¹³ is H, or, if,

E is NH₂, and F, R¹³, U', U" and U'" are OH, then e and/or f is/are 0, or, if

E is NH₂, and F, R¹³, U" and U"' are OH, and e and f are 1, then U' is mercapto.

The novel compounds of the formula VI can be employed as aids in molecular biology, for example in PCR reactions (e=f=1, R¹³ =OH) or for sequencing (e=f=1; R¹³ =H or OH).

The compounds of the formula VI can be prepared from the corresponding 8-azapurine nucleosides using generally known methods. The compounds of the formula VI may preferably be prepared using an abbreviated version of Ludwig's one pot process in the presence of 1,8-bis(dimethylamino)naphthalene and trimethyl phosphate J. Ludwig et al., (1981) Acta Biochem. Biophys. Sci. Hung., 16, 131!.

The invention also embraces all the tautomeric forms of the compounds of the formulae I, V and VI, in particular all the tautomeric forms of the 8-azapurine bases of the formula IV.

The invention furthermore relates to a process for preparing the novel compounds of the formula I.

Grunberger et al. Biochim. Biophys. Acta, (1968) 161, 147-155! have described the preparation of triribonucleoside diphosphates containing 8-azaguanine by an enzymic or chemoenzymic route. Bodnar et al. describe the enzymic synthesis of double-stranded, 8-azaguanine-containing phage DNA using DNA polymerase Bodnar et al., (1983) J. Biol. Chem., 258, 15206-15213!.

Owing to the fact that the N-glycosidic bond of 8-azadeoxyguanosine is extremely stable to acid Seela et al., (1993), Helv. Chim. Acta, 76, 2388-2397!, the standard conditions which are customary in the chemical synthesis of oligonucleotides may be employed for preparing the novel 8-azapurine-containing oligonucleotides.

The novel compounds of the formula I are prepared in solution or, preferably, on a solid phase, where appropriate using an apparatus for automatic synthesis. The construction of the oligomers of formula I can take place stepwise by condensing mononucleotides, in each case containing a nucleotide base, one at a time, onto an appropriately derivatized support or onto a growing oligomer chain.

The oligonucleotide is constructed using the methods known to the person skilled in the art, such as the triester method, the H-phosphonate method or the phosphoramidite method E. Sonveaux, (1986), Bioorganic Chemistry, 14, 274-325; S. L. Beaucage et al., (1992), Tetrahedron, 48, 2223-2311!. The nucleotide monomer building blocks of the formula V are preferably employed for introducing the 8-azapurine derivatives, with those nucleotide monomers of the formula V in which E' is NR¹⁰ R¹¹ and F' is OH being particularly preferred.

The compounds of the formula V, as building blocks for the oligonucleotide solid phase synthesis, can be prepared from the corresponding 8-azapurine nucleosides. Once appropriate protective groups for the amino groups of the 8-azapurine bases and the free 5'-hydroxyl group of the sugar have been introduced, the monomers are converted into the corresponding phosphonate or phosphoramidite derivatives. Suitable amino protective groups, for example in the form of a formamidine protective group ((dimethylamino)methylidene) or acyl protective group, are introduced using generally known methods L. J. McBride et al, (1983) Tetrahedron Lett., 24, 2953, G. S. Ti et al, (1982) J. Am. Chem. Soc., 104, 1316; H. Schaller et al. (1963), J. Am. Chem. Soc., 85, 3821!, with the use of Schaller's peracylation method being advantageous when the amino group is acylated. An example of a suitable protective group for the free 5'-OH group of the sugar is 4,4'-dimethoxytrityl, which is likewise introduced using known methods C. B. Reese (1978), Tetrahedron, 34, 3143; D. Flockerzi et al., (1981), Leibigs Ann. Chem., 1568!. The monomers which have been protected in this way can be converted to the corresponding phosphonates using a protocol developed by Froehler et al., B. C. Froehler et al., (1986), Nucl. Acid Res., 14, 5399!. Cyanoethylphophoramidite derivatives can be prepared, for example, by reacting the monomers with chloro-β-cyanoethoxy(N,N-diisopropylamino)phosphane in anhydrous dichloromethane N. D. Sinha et al., (1984) Nucl. Acid Res., 12, 4539!.

The oligoribonucleotide synthesis is rendered more difficult, as compared with the deoxyribooligonucleotide synthesis, as a result of the additional 2'-OH group. The first difficulty is that of finding a combination of compatible 2'-OH and 5'-OH protective groups. Thus, during the oligonucleotide synthesis, the residue at the O-2' position has to be stable towards the acid conditions used for hydrolysing the trityl protective groups. In addition, conditions have to be avoided which could lead to migration of the phosphate radical from the 3' position to the 2' position. A regioselective reaction is desirable when introducing the protective group.

It is advantageous for synthesizing the novel oligoribonucleotides of the formula (I) to use triisopropylsilyl chloride as the 2'-OH protective group, with which high degrees of selectivity are achieved in association with adequate stability and mild elimination conditions (TBAF/THF). The selectivity of the reaction can be increased still further if silver nitrate is used as the catalyst in place of imidazole F. Seela, K. Mersmann, J. A. Grasby, M. J. Gait, Helv. Chim. Acta 1993, 76, 1809!.

In contrast to the situation as regards the solid phase synthesis of deoxyribonucleotides, phosphoramidite building blocks are not particularly suitable for the solid phase synthesis of oligoribonucleotides. This is due, in particular, to relatively long coupling times being required as a result of the reactive phosphoramidite being sterically hindered by the bulky 2'-silyl protective group N. Usman, R. T. Pon, K. K. Ogilvie, Tetrahedron Lett. 1985, 26, 4567!, which hinderance also results in a lower coupling yield. Ribonucleoside phosphonates are more stable towards hydrolysis and oxidation than are the phosphoramidites and permit the oligonucleotide synthesis to take place with relatively short cycling times.

The ribonucleotide phosphonates according to the invention may be prepared using a protocol developed by Froehler B. Froehler, P. G. Nug, M. D. Mateuci, Nucl. Acids Res. 1986, 14, 5399!.

Compounds of the formula I whose oligonucleotide moieties are modified at the 3' and/or 5' ends are synthesized, with respect to these modifications, using the methods described in EP-A 0 552 766.

The invention also relates to the use of the novel compounds of the formula I for preparing a pharmaceutical, and also to a process for preparing a pharmaceutical which comprises mixing the novel oligonucleotides with a physiologically acceptable excipient and also, where appropriate, suitable additives and/or auxiliary substances.

In a quite general manner, the present invention extends to the use of compounds of the formula I as therapeutically active components of a pharmaceutical. In general, therapeutically active oligonucleotide derivatives are understood to mean antisense oligonucleotides, triple helix-forming oligonucleotides, aptamers or ribosomes, in particular antisense oligonucleotides.

In addition to this, the present invention also relates to the use of oligonucleotides containing at least one 8-azapurine, preferably containing 8-azaguanine or 8-azaadenine, as a diagnostic agent, for example for detecting the presence or absence, or the quantity, of a specific double-stranded or single-stranded nucleic acid molecule in a biological sample.

For being used in accordance with the invention, the oligonucleotides have a length of from 4 to 100, preferably of approximately 5-40, in particular of approximately 6-30, nucleotides. Apart from that, the abovedescribed preference ranges, modifications and conjugations also apply in this instance.

The pharmaceuticals of the present invention can be used, for example, for treating diseases which are elicited by viruses, for example by HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.

Novel antisense oligonucleotide derivatives, that is antisense oligonucleotides in which at least one purine base is replaced by an 8-azapurine base, and which are active against targets of this nature, have the following base sequences, for example:

a) against HIV, e.g.

    5'-A C A C C C A A T T C T G A A A A T G G-3' (SEQ. ID. No.1)(I)

    or

    5-A G G T C C C T G T T C G G G C G C C A-3' (SEQ. ID. No.2)(II)

    or

    5'-G T C G A C A C C C A A T T C T G A A A A T G G A T A A-3' (SEQ. ID. No.3)                                                     (III)

    or

    5'-G C T A T G T C G A C A C C C A A T T C T G A A A-3' (SEQ. ID. No.4)(IV)

    or

    5'-C T G T C T C C G C T T C T T C T T C C T G C C A T A G G A G (SEQ. ID. No.6)                                                     (V)

    or

    5-T C G T C G C T G T C T C C G C T T C T T C T T C C T G C C A (SEQ. ID. No.5)                                                     (VI)

    or

b) against HSV-1, e.g.

    5'-G C G G G G C T C C A T G G G G G T C G-3' (SEQ. ID. No.7)(VII)

The pharmaceutials of the present invention are also suitable for treating cancer, for example. For example, oligonucleotide sequences can be employed in this context which are directed against targets which are responsible for the appearance and growth of cancers. Examples of such targets are:

1) Nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA and p120

2) Cytoplasmic/membrane-associated oncoproteins such as, for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src and c-abl

3) Cellular receptors, such as, for example, EGF receptor, c-erbA, retinoid receptors, protein kinase regulatory subunit and c-fms

4) Cytokines, growth factors and extracellular matrix, such as, for example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, bFGF, myeloblastin and fibronectin.

Novel antisense oligonucleotides of the formula I which are active against targets of this nature have the following base sequences, for example:

a) against c-Ha-ras, e.g.

    5'-C A G C T G C A A C C C A G C-3' (SEQ. ID. No.8)        (VIII)

c) c-myc, e.g.

    5'-G G C T G C T G G A G C G G G G C A C A C-3' (SEQ. ID. No.9)(IX)

    5'-A A C G T T G A G G G G C A T-3' (SEQ. ID. No.10)

d) c-myb, e.g.

    5'-G T G C C G G G G T C T T C G G G C-3' (SEQ. ID. No.12) (XI)

e) c-fos, e.g.

    5'-G G A G A A C A T C A T G G T C G A A A G-3' (SEQ. ID. No.14)(XII)

    5'-C C C G A G A A C A T C A T G G T C G A A G-3' (SEQ. ID. No.15)(XIII)

    5'-G G G G A A A G C C C G G C A A G G G G-3' (SEQ. ID. No.16)XIV)

f) p120, e.g.

    5'-C A C C C G C C T T G G C C T C C C A C-3' (SEQ. ID. No.17)(XV)

g) EGF receptor, e.g.

    5'-G G G A C T C C G G C G C A G C G C-3' (SEQ. ID. No.18) (XVI)

    5'-G G C A A A C T T T C T T T T C C T C C-3' (SEQ. ID. No.19)(XVII)

h) p53 tumor suppressor, e.g.

    5'-G G G A A G G A G G A G G A T G A G G-3' (SEQ. ID. No.20)(XVIII)

    5'-G G C A G T C A T C C A G C T T C G G A G-3' (SEQ. ID. No.21)(XIX)

The pharmaceuticals of the present invention are also suitable, for example, for treating diseases which are affected by integrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM, VCAM or ELAM.

Novel antisense oligonucleotide derivatives which are active against targets of this nature have the following base sequences, for example:

a) VLA-4, e.g.

    5'-G C A G T A A G C A T C C A T A T C-3' (SEQ. ID. No.24) (XX)

    or

b) ICAM, e.g.

    5'-C C C C C A C C A C T T C C C C T C T C-3' (SEQ. ID. No.25)(XXI)

    5'-C T C C C C C A C C A C T T C C C C T C-3' (SEQ. ID. No.26)(XXII)

    5'-G C T G G G A G C C A T A G C G A G G-3' (SEQ. ID. No.27)(XXIII)

c) ELAM-1, e.g.

    5'-A C T G C T G C C T C T T G T C T C A G G-3' (SEQ. ID. No.28)(XXIV)

    5'-C A A T C A A T G A C T T C A A G A G T T C-3' (SEQ. ID. No.29)(XXV)

The pharmaceuticals of the present invention are also suitable, for example, for preventing restenosis. For example, oligonucleotide sequences may be employed in this context which are directed against targets which are responsible for proliferation or migration. The examples of targets of this nature are:

1) Nuclear transactivating proteins and cyclines, such as, for example, c-myc, c-myb, c-fos, c-fos/jun, cyclines and cdc2 kinase,

2) Mitogens or growth factors, such as, for example, PDGF, bFGF, EGF, HB-EGF and TGF-β.

3) Cellular receptors, such as, for example, bFGF receptor, EGF receptor and PDGF receptor.

Novel oligonucleotides of the formula I which are active against targets of this nature have the following base sequences, for example:

a) c-myb

    5'-G T G T C G G G G T C T C C G G G C-3' (SEQ. ID. No.13) (XXVI)

b) c-myc

    5'-C A C G T T G A G G G G C A T-3' (SEQ. ID. No.11)       (XXVII)

c) cdc2 kinase

    5'-G T C T T C C A T A G T T A C T C A-3' (SEQ. ID. No.22) (XXVIII)

d) PCNA (proliferating cell nuclear antigen of rat)

    5'-G A T C A G G C G T G C C T C A A A-3' (SEQ. ID. No.23) (XXIX)

The pharmaceuticals may, for example, be used in the form of pharmaceutical preparations which can be administered orally, for example in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions or suspensions. Enclosure of the pharmaceuticals in liposomes, which; where appropriate, contain additional components such as proteins, also represents a suitable form of administration. They may also be administered rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions. In order to produce pharmaceutical preparations, these compounds can be processed in therapeutically inert organic and inorganic excipients. Examples of excipients of this nature which are suitable for tablets, coated tablets and hard gelatin capsules are lactose, corn starch or derivatives thereof, tallow and stearic acid or salts thereof. Water, polyols, sucrose, invert sugar and glucose are suitable excipients for preparing solutions. Water, alcohols, polyols, glycerol and vegetable oils are suitable excipients for injection solutions. Vegetable and hardened oils, waxes, fats and semiliquid polyols are suitable excipients for suppositories. The pharmaceutical preparations may also contain preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for altering the osmotic pressure, buffers, coating agents and antioxidants, and also, where appropriate, additional therapeutic active compounds.

Preferred forms of administration are topical administration, local administration, for example using a catheter, and also injections. For an injection, the antisense oligonucleotide derivatives are formulated in a liquid solution, preferably in a physiologically acceptable buffer, such as, for example, Hank's solution or Ringer's solution. However, the antisense oligonucleotides can also be formulated in solid form, and dissolved or suspended prior to use. The dosages which are preferred for systematic administration are from approximately 0.01 mg/kg to approximately 50 mg/kg of body weight and per day.

In a quite general manner, the invention extends to the use of compounds of the formula I as DNA probes or primers in DNA diagnostics, and generally as aids in molecular biology.

EXAMPLES

The compounds (1)-(16), which are mentioned in the examples, have the following structural formulae.

    ______________________________________                                          ##STR11##                                                                      ##STR12##                                                                     ______________________________________                                          ##STR13##                                                                              R                                                                     ______________________________________                                                   ##STR14##                                                            5                                                                                        ##STR15##                                                            6                                                                                        ##STR16##                                                            ______________________________________                                          ##STR17##                                                                          R(a)     R(b)      R(c)   R                                               ______________________________________                                          (7) COCH.sub.3                                                                              COCH.sub.3                                                                               COCH.sub.3                                                                            NHCOC.sub.5 H.sub.19                             (8) COCH.sub.3                                                                              COCH.sub.3                                                                               COCH.sub.3                                                                            NH.sub.2                                         (9) OH       OH        OH     NH.sub.2                                        (10) OH       OH        OH     NHCOC.sub.6 H.sub.5                             (11) OH       OH        OH     NCHN(CH.sub.3).sub.2                            (12) OH       OH        OH                                                                                     ##STR18##                                      (13) OH       OH        Dmt    "                                               (14) Tms      OH        Dmt    "                                               (15) OH       Tms       Dmt    "                                               (16) Tms      TEP       Dmt    "                                               ______________________________________                                    

Example 1

5-Amino-3-(2-deoxy-β-D-erythropentofuranosyl)-3H-1,2,3triazolo 4,5-d!pyrimidin-7-(6H)-one-5'-O-triphosphate, triethylammonium salt (8-aza-2'-deoxyguanosine 5'-triphosphate)

While warming gently, 3-(2-deoxy-β-D-erythropentofuranosyl)-5-amino-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one (8-aza-2'-deoxyguanosine (1)) (26 mg; 0.09 mmol) was brought, together with 1,8-bis(dimethylamino)naphthalene (33 mg, 0.15 mmol), into solution in trimethyl phosphate (0.25 ml). After the solution had been cooled down to 0° C., freshly distilled POCl₃ (12 μl, 0.13 mmol) was added. The reaction was maintained at 4° C. for 4 h, and a solution composed of tri-n-butylammonium diphosphate (0.5 mM in DMF, 1 ml) and tri-n-butylamine (100 μl, 0.42 mmol) was then added. After this mixture had been stirred at 0° C. for 3 min, 1M TBC buffer (10 ml) was added and the whole was evaporated to dryness. The residue was chromatographed on DEAD Sephadex® (1.5×20 cm column, HCO₃ ⁻ form). After the column had been washed with approximately 500 ml of H₂ O, chromatography took place using a linear gradient of H₂ O/0.9M TBC buffer (1 l in each case). This yielded a main zone at approximately 0.5M TBC buffer (0.019 mM, 20%).

TLC (silica gel, i-propanol/H₂ O/NH₃, 3:1:1): R_(f) 0.2. UV(H₂ O): λ_(max) 256 nm.

³¹ P-NMR (0.1M Tris-HCl, pH 8.0, 100 mM EDTA/D₂ O): -10.27 (d,J=19.3, P_(x)); -10.63 (td, J=20.2 and 6.0, P.sub.α); -22.60 (t, J=19.8, P.sub.β).

Example 2

3-(2-Deoxy-β-D-erythropentofuranosyl)-5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one (2).

3-(2-Deoxy-β-D-erythropentofuranosyl)-5-amino-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one (8-aza-2'-deoxyguanosine (1)) (290 mg; 1.06 mmol) was dissolved in abs. DMF (7 ml), and N,N-dimethylformamide diethyl acetal (5 ml) was added. After the mixture had been stirred at room temperature for 24 hours, it was subjected to rotary evaporation in vacuo, and the residue was coevaporated with toluene. The residue was purified by means of chromatography on silica gel (column: 20×4 cm, 0.5 bar, CH₂ Cl₂ /MeOH 8:2). Compound (2) (320 mg, 92%) was obtained as a colorless foam which crystallized from MeOH. M.p. 236° C. TLC (CH₂ Cl₂ /MeOH 8:2): R_(f) 0.7. UV (MeOH): 301 (26500), 244(15500).

¹ H-NMR ((D₆)DMSO: 8.66 (s, CH); 6.43 (t, J=6.32, H--C(1')); 5.40 (br.s, OH--C(3')); 4.79 (br.s, OH--C(5')), 4.48 (m, H--C(3')); 3.85 (m, H--C(4')); 3.57 (m, H--C(5')); 3.19, 3.06 (2 s, 2CH₃); 2.90 (m, H.sub.β --C(2')); 2.34 (m, H.sub.(α) --C(2')).

C₁₂ H₁₇ N₇ O₄ (323.31) calculated C 44.56, H 5.29, N 30.32; found C 44.64, H 5.26, n 30.37.

Example 3

3- 2-Deoxy-5-O-(4,4'-dimethoxytrityl-β-D-erythropentofuranosyl!-5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one (3).

The amino-protected 8-aza-2'-deoxyguanosine (2) from Example 2 (170 mg, 0.53 mmol) was treated by being repeatedly coevaporated with abs. pyridine and then taken up in 6 ml of the latter. 4,4'-Dimethoxytrityl chloride (260 mg, 0.7 mmol) was added at room temperature, and this mixture was stirred for 3 hours. The solution was then poured into 5% NaHCO₃ (40 ml), and this mixture was extracted twice with 30 ml of CH₂ Cl₂ on each occasion. The combined organic phases were dried over Na₂ SO₄ and subjected to rotary evaporation in vacuo. The residue was chromatographed on silica gel (column: 15×4 cm, 0.5 bar, CH₂ Cl₂ /MeOH 95:5). 270 mg (82%) of a colorless foam were obtained.

TLC (CH₂ Cl₂ /MeOH 95:5) R_(f) 0.3. UV (MeOH): 302 (24600), 235 (35000).

¹ H-NMR ((D₆)DMSO): 12.03 (s, NH); 8.66 (s, CH); 7.39 -6.70 (2 m, aromat. H); 6.48 (m, H--C(1')); 4.57 (m, H--C(3')); 3.97 (m, H--C(4')); 3.69 (s, 2OCH₃); 3.37 (m, H--C(5')), 3.22, 3.07 (2s, 2CH₃); 2.90 (m, H.sub.β --C(2')); 2.40 (m, H.sub.(α) --C(2')).

C₃₃ H₃₅ N₇ O₆ (625.68) calculated C 63.34, H 5.64, N 15.67; found C 63.42, H 5.72, N 15.71.

Example 4

3- 2-Deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!-5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'(triethylammonium phosphonate) (4).

1,2,4-Triazole (0.54 g, 7.56 mmol) was added to a solution of PCl₃ (200 μl, 0.22 mmol) and N-methylmorpholine (2.7 ml, 2.24 mmol) in CH₂ Cl₂ (10 ml). The solution was left to stir for 30 min and then cooled down to 0° C. The 5'-O-dimethoxytrityl compound 3 from Example 3 (220 mg, 0.35 mmol) was dried by coevaporation with dried MeCN and was then added after being dissolved in CH₂ Cl₂ (5 ml). After it had been stirred for 10 min, the mixture was poured into 1M (Et₃ NH)HCO₃ (TBC buffer, pH 8.0, 30 ml), and this latter mixture was extracted twice by shaking with CH₂ Cl₂ and separating the phases. The combined organic solutions were dried over Na₂ SO₄ and evaporated. The residue was chromatographed on silica gel (column: 4×15 cm, 0.5 bar; 1. CH₂ Cl₂ /Et₃ N 98:2; 2. CH₂ Cl₂ /MeOH/Et₃ N 88:10:2). The substance present in the main zone was taken up in CH₂ Cl₂ (10 ml), and this solution was extracted several times by shaking with 0.1M TBC buffer (pH 8.0). The H-phosphonate 4 (240 mg, 85%) was obtained as a colorless foam. TLC (CH₂ Cl₂ /MeOH/Et₃ N 88:10:2): R_(f) 0.3. UV (MeOH): 285 (sh, 14900), 303 (18400).

¹ H-NMR ((D₆)DMSO): 11.68 (s, NH); 8.83 (s, CH); 7.77, 5.45, (d, J=585, HP); 7.24-6.69 (2 m, aromat. H); 6.53 (m, J=4.5, H--Cl')); 5.22 (m, H--C(3')); 4.09 (m, H--C(4')); 3.67 (s, 2 OCH₃); 3.07, 3.23 (s, 2 CH₃, H--C(5')); 2.97 (m, CH₃ CH₂); 2.56 (m, H--C(2')); 1.13 (m, CH₃ CH₂).

³¹ P-NMR ((D₆)DMSO): 1.16 (¹ J(P,H)=585); ³ J(P,H--C(3')=8.90.

C₃₉ H₅₁ N₈ O₈ P₁ (790.87) calculated C 59.23, H 6.49, N 14.17; found C 59.33, H 6.79, N 13.95.

Example 5

3- 2-Deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!-5-{(dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'- (2-cyanoethyl)-N,N-diisopropylphosphoramidite! (5).

(i-Pr)₂ EtN (56 μl, 0.27 mmol) and chloro(2-cyanoethoxy)diisopropylaminophosphane (115 μl, 0.51 mmol) were added to a solution of compound (3) from Example 3 (50 mg, 0.08 mmol) in CH₂ Cl₂ (1 ml). The mixture was stirred at room temperature for 2 hours under argon. It was then poured into 5% NaHCO₃ (3 ml), and this mixture was extracted twice with CH₂ Cl₂ (30 ml). The combined organic phases were dried over Na₂ SO₄ and evaporated to dryness on a rotary evaporator. The residue was chromatographed on silica gel (column: 7×2 cm, 0.5 bar, CH₂ Cl₂ /AcOEt/Et₃ N 45:45:10). It was possible to distinguish two overlapping zones of diastereomers of the phosphoramidite (5), which diastereomers were obtained as a colorless foam (35 mg, 55% ).

TLC (CH₂ Cl₂ /AcOET/Et₃ N 45:45:10): R_(f) 0.4.

³¹ P-NMR ((D₆)DMSO): 149.4, 148.9.

Example 6

2- 2-Deoxy-5-O-(4,4-dimethoxytrityl)-3'-O-succinyl-β-D-erythropentofuranosyl!-5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one (6).

4-Dimethylaminopyridine (30 mg, 0.23 mmol) and succinic anhydride (90 mg, 0.88 mmol) were added to a solution of the protected nucleoside (3) from Example 3 (110 mg, 0.18 mmol) in pyridine (5 ml). The mixture was left to stir at room temperature for 48 hours. The reaction was stopped by adding 2 ml of water. After the mixture had been evaported to dryness, the residue was coevaporated with toluene in order to remove any remaining pyridine. The residue was dissolved in a little CH₂ Cl₂, and this solution was washed with a 10% aqueous solution of citric acid and with water. The organic phase was dried over Na₂ SO₄ and concentrated in vacuo. After the residue had been dissolved in CH₂ Cl₂ /pyridine (95:5, 2 ml), n-pentane/ether (1:1, 30 ml) was added rapidly. The supernatant was filtered off and a colorless powder remained (85 mg, 66%). TLC (CH₂ Cl₂ /MeOH 9:1): R_(f) 0.25. ¹ H-NMR ((D₆)DMSO): 8.71 (s, CH); 7.28-6.75 (2 m, aromat. H); 6.46 (m, J=4.1, H--C(1')); 5.48 (m, H--C(3')); 4.21 (m, H--C(4')); 3.36 (s, 2 OCH₃); 3.18, 3.05 (2 s, 2 CH₃); 3.10 (m, H--C(5')); 2.51 (m, H--C(2'), 2 CH₂).

Example 7

7-(Benzoylamino)-1,2,3-triazolo 4,5-d!pyrimidine

8-Azaadenine (200 mg, 1.47 mmol) is evaporated three times in dry pyridine and then taken up in 5 ml of dry pyridine. Benzoyl chloride (0.28 ml, 2.20 mmol) is then added dropwise, and the mixture is stirred at 60° C. for 3 hours. It is then boiled under reflux for a further hour. The reaction mixture is left to stand overnight and is then concentrated down to approximately 1 ml. 15 ml of cold water are added to this mixture, which is left to stir for 5 min; the resulting precipitate is filtered off with suction. The faintly yellowish precipitate is washed twice in each case with 1 ml of cold water and 1 ml of cold acetonitrile. 0.30 g (85%) of colorless crystals are obtained (MeOH). M.p.=263° C. (decomposition). TLC (silica gel, CH₂ Cl₂ /MeOH=8:2); R_(f) =0.6. UV (methanol) λ_(max) (ε)=242 (12100); 291 (16000). ¹ H-NMR (D₆ -DMSO) δ: 7.58; 7.69; 8.13 (arom. --H₅) 8.89 (s, H-5), 11.99 (s-N⁶ --H). C₁₁ H₈ N₆ O calculated C 54.99 H 3.36 N 34.99. found C 55.10 H 3.34 N 35.04.

Example 8

7-(Nonanoylamino)-3- (2,3,5-tri-O-acetyl)-β-D-ribofuranosyl!-3H-1,2,3-triazolo 4,5-d!pyrimidine (7)

500 mg (1.81 mmol) of 7-nonanoylamido-1,2,3-triazolo 4,5d!pyrimidine are evaporated to dryness three times in dry pyridine. The residue is taken up in 20 ml of dry acetonitrile. 0.58 g (1.81 mmol) of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose is added, 0.64 ml (5.43 mmol) of tin tetrachloride is added dropwise, and the reaction mixture is stirred at room temperature for 24 hours. The reaction solution is then added to 25 ml of a saturated solution of sodium hydrogen carbonate, and this mixture is extracted four times with 15 ml of dichloromethane on each occasion. After having been dried over sodium sulfate, the combined organic phases are evaporated to dryness. 0.81 g of an oily residue is obtained. The product mixture is fractionated by column chromatography (5.5×30 cm column, silica gel, eluent: CH₂ Cl₂ /MeOH 95:5). 0.27 g (28%) of a colorless nucleoside is obtained from the more rapidly migrating zone. TLC (silica gel, CH₂ Cl₂ /MeOH): R_(f) 0.75. UV (methanol) λ_(max) (ε)=275 (16800). ¹ H-NMR (D₆ -DMSO) δ: 0.82 (m, --CH₃ -9"); 1.22 (m, --(CH₂)₅ --); 1.62 (m, J=6.8 Hz, --CH₂ -3"); 1.93; 2.08; 2.11 (3s, O═CCH₃); 2.61 (t, J=7.2 Hz, --CH₂ -2"); 4.26 (m, H-5'); 4.52 (m, H-4'); 5.77 (m, H-3'); 6.12 (m, H-2'); 6.63 (d, J=3.4 Hz, H-1'); 8.89 (s, H-5); 11.48 (s, br, N⁶ --H).

Example 9

Glycosylation of 7-amino-1,2,3-triazolo 4,5-d!pyrimidine with 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose.

340 mg (2.5 mmol) of 7-amino-1,2,3-triazolo 4,5-d!pyrimidine and 800 mg (2.5 mmol) of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose are suspended in 10 ml of dry acetonitrile. 0.88 ml (7.5 mmol) of tin tetrachloride is added dropwise, under argon, to this mixture within the space of 5 min, and the whole is then stirred at room temperature for 24 hours. The resulting solution is poured carefully onto 32 ml of a saturated solution of NaHCO₃. The precipitate which arises is filtered off with suction and washed twice with 10 ml of water on each occasion. The filtrate and the washing water are extracted four times with 15 ml of methylene chloride on each occasion. After drying over Na₂ SO₄, and stripping off the solvent, 0.87 g of a faintly yellowish foam is obtained. The reaction products are fractionated by column chromatography (column: 5.5×20 cm, silica gel, CH₂ Cl₂ /MeOH 98:2-90:10).

Example 10

7-Amino-3- (2,3,5-tri-O-acetyl)-β-D-ribofuranosyl!-3H-1,2,3-triazolo 4,5-d!pyrimidine (8)

The more rapidly migrating zone yields 0.34 g (34%) of a colorless foam.

TLC (silica gel, CH₂ Cl₂ /MeOH 9:1): R_(f) =0.45. UV (methanol) λ_(max) (ε)=280 (10900). ¹ H-NMR (D₆ -DMSO) δ: 1.89; 2.10; 2.11 (3s, 2',3',5'-OC═O); 4.20 (m, H₂ -5'); 4.48 (m, H-4'); 5.74 (t, J=5.5 Hz, H-3'); 6.07 (t, J=3.7 Hz, H-2'); 6.48 (d, J=2.8, H-1'); 8.25; 8.6 (2s, N⁶ --H₂), 8.34 (s, H-5).

C₁₅ H₁₈ N₆ O₇ calculated C 45.68 H 4.61 N 21.31; found C 45.98 H 4.72 N 21.40.

Example 11

7-Amino-2- (2,3,5-tri-O-acetyl)-β-D-ribofuranosyl!-2H-1,2,3-triazolo 4,5-d!pyrimidine

0.46 g (47%) of a colorless foam are obtained from the more slowly migrating zone of the chromatographic purification step.

TLC (silica gel, CH₂ Cl₂ /MeOH): R_(f) =0.35 UV (methanol) λ_(max) (ε)=253 (3900), 300 (10400). ¹ H-NMR (D₆ -DMSO) δ: 1.94; 2.07; 2.11 (3s, 2',3',5'-OC═O); 4.25 (m, H₂ -5'); 4.52 (m, H-4'); 5.74 (t, J=6.0, H-3'); 5.91 (d, J=3.4, H-2'); 6.53 (s, H-1'); 8.3; 8.45 (2s, N⁶ --H₂); 8.34 (H-5). C₁₅ H₁₈ N₆ O₇ calculated C 45.68 H 4.61 N 21.31; found C 45.86 H 4.71 N 21.22.

Example 12

7-Amino-3(β-D-ribofuranosyl)-3H-1,2,3-triazolo 4,5d!pyrimidine (8-azaadenosine, 9)

2.04 g (5.17 mmol) of compound (8) are stirred, at room temperature for 2 hours, in 5 ml of methanol and 5 ml of aqueous ammonia (25%). After evaporation to dryness and recrystallization of the residue in 3 ml of water, 1.04 g (75%) of colorless crystals are obtained which decompose at 217° C.

TLC (silica gel, CH₂ Cl₂ /MeOH=8:2): R_(f) =0.45. UV: λ_(max) (pH 7)=279 (11600); λ_(max) (pH 1)=263, λ_(max) (pH 14)=279. ¹ H-NMR (D₆ -DMSO) δ: 3.56 (m, H₂ -5'); 4.01 (m, H-4'), 4.29 (m, 3'); 4.85 (m, H-2'); 5.01 (t, J=5.9 Hz, HO-5'); 5.28 (d, J=5.7, HO-3'); 5.56 (d, J=6.0 HO-2'); 6.15 (d, J=5.2 Hz, H-1'); 8.31 (s, H-5); 8.53; 8.19 (2s, N⁷ H₂).

Example 13

7-Amino-2-(β-D-ribofuranosyl)-2H-1,2,3-triazolo 4,5d!pyrimidine

0.81 g (2.05 mmol) of the compound from Example 11 is stirred, at room temperature for 2 hours, in 5 ml of methanol and 5 ml of aqueous ammonia (25%). After evaporation to dryness and recrystallization of the residue in 2.5 ml of water, 0.32 g (59%) of colorless crystals is obtained, which crystals decompose at 209° C.

TLC (CH₂ Cl₂ /MeOH=8:2): R_(f) =0.20 UV (methanol) λ_(max) (ε)=255 (4400), 263 (4100), 297 (10400). ¹ H-NMR (D₆ -DMSO) δ: 3.59 (m, H2-5'); 4.06 (m, H-4'); 4.33 (m, H-3'); 4.61 (m, H-2'); 4.76 (t, J=5.3 Hz, HO-5'); 5.27 (d, J=5.8 Hz, HO-3'); 5.68 (d, J=5.5 Hz, HO-2'); 6.08 (s, J=3.4 Hz, H-1'); 8.31 (s, H-5); 8.12 (s, N⁷ H₂).

Example 14

7-Benzoylamino-3-(β-D-ribofuranosyl)-3H-1,2,3-triazolo 4,5-d!pyrimidine (10).

100 mg (0.37 mmol) of 7-amino-3-β-D-ribofuranosyl-3H-1,2,3-triazolo 4,5-d!pyrimidine are initially introduced in 5 ml of dry pyridine. 0.47 ml (3.7 mmol) of trimethylsilyl chloride is added dropwise to this solution under an argon atmosphere. After the mixture has been stirred at room temperature for half an hour (monitoring by TLC), 0.25 ml (2.0 mmol) of benzoyl chloride is added dropwise, and this reaction mixture is stirred at room temperature for 4 hours. It is then cooled down to 0°-5° C. and 1 ml of water is added followed, 5 min later, by 2 ml of aqueous ammonia (25%); this mixture is then stirred for a further 30 min. The solvent is stripped off and the residue is then evaporated with toluene. The residue is taken up in 15 ml of a saturated solution of NaHCO₃, and this mixture is extracted once with 25 ml of methylene chloride and several times with ethyl acetate. The combined organic phases are dried using Na₂ SO₄.

After the solvent has been stripped off, 0.05 g (36%) is obtained of a colorless, crystalline substance which, after crystallization from methanol, melts with decomposition at 188° C.

TLC (silica gel, CH₂ Cl₂ /MeOH=9:1): R_(f) =0.25. UV (methanol) λ_(max) (ε)=242 (9400), 282 (20300). ¹ H-NMR (D₆ -DMSO) δ: 3.56 (m, H₂ -5'); 4.04 (m, H-4'); 4.36 (m, H-3'); 4.84 (t, HO-5'); 4.92 (m, H-2'); 5.33 (d, J=5.2 Hz, HO-3'); 5.66 (d, J=5.4 Hz, HO-2'); 6.30 (d, J=4.3 Hz, 1'); 7.54-8.11 (m, arom.--H₅); 8.94 (s, H-5); 11.99 (s, br, N⁶ --H). C₁₆ H₁₆ N₆ O₆ calculated C 51.60 H 4.34 N 22.57. found C 51.49 H 4.43 N 22.74.

Example 15

7-{ (Dimethylamino)methylidene!amino}-3-(β-D-ribofuranosyl)-3H-1,2,3-triazolo 4,5-d!pyrimidine (11).

100 mg (0.37 mmol) of 7-amino-3-β-D-ribofuranosyl-(3H)-1,2,3-triazolo 4,5-d!pyrimidine are left to stir overnight, at room temperature, in 2 ml of dry DMF and 0.25 ml (1.85 mmol) of N,N-dimethylformamide dimethyl acetal. 4 ml of methanol is then added to this reaction mixture. After having been stirred for a further 2 hours, the mixture is evaporated to dryness, and the residue is coevaporated with toluene and then chromatographed on silica gel. (Column: 3×20 cm, eluent CH₂ Cl₂ /MeOH 98:2-90:10). 0.06 g (52%) of a colorless, glassy solidified substance is obtained in the main zone.

TLC (silica gel, CH₂ Cl₂ /MeOH=9:1): R_(f) =0.25. UV λ_(max) (ε)=235, 325. ¹ H-NMR (D₆ -DMSO) δ: 3.21; 3.27 (2s, N (CH₃)₂); 3.55 (m, H₂ -5'), 4.00 (m, H-4'); 4.31 (m, H-3'); 4.87 (m, H-2'); 4.96 (t, HO-5'); 5.32 (d, HO-3'); 5.60 (d, HO-2'); 6.19 (d, J=4.7 Hz, H-1'); 8.57 (s, H-5); 9.06 (s, N=CH).

Example 16

7-{ 1-(Dimethylamino)ethylidene!amino}-3-(6-D-ribofuranosyl)-3H-1,2,3-triazolo 4,5-d!pyrimidine (12).

0.91 ml (5.59 mmol) of N,N-dimethylacetamide dimethyl acetal is added to 500 mg (1.86 mmol) of 9 in 10 ml of analytical grade methanol. The suspension is left to stir at room temperature for 14 hours. The solution which results is freed from the solvent on a rotary evaporator, and the residue is coevaporated with toluene. A further 10 ml of methanol are added to the residue, and this mixture is stirred at room temperature for 2 hours. After the solvent has been separated off, the residue is chromatographed on silica gel (column, 4×20 cm, eluent CH₂ Cl₂ /MeOH=98:2-90:10). Yield: 0.48 g (76%) of a colorless foam.

TLC (silica gel, CH₂ Cl₂ /MeOH=9:1): R_(f) =0.35. UV (methanol) λ_(max) (ε)=233 (9300), 270 (3600), 324 (26100). ¹ H-NMR (D₆ -DMSO) δ: 2.28 (s, N=C--CH₃); 3.20 (s, N(CH₃)₂); 3.55 (m, H₂ -5'); 4.00 (m, H-4'); 4.30 (m, H-3'); 4.86 (m, H-2'); 4.98 (t, HO-5'); 5.29 (d, J=5.2 Hz, HO-3'); 5.57 (d, J=5.9 Hz, HO-2'), 6.18 (d, J=5.2 hZ, H-1'); 8.54 (S, H-5). C₁₃ H₁₉ N₇ O₄ calculated C 46.28 H 5.69 N 29.07. found C 46.45 H 5.63 N 28.97.

Example 17

7-{ 1-(Dimethylamino)ethylidene!amino}-3- 5-O-(4,4'-dimethoxytriphenylmethyl)-β-D-ribofuranosyl!-3H-1,2,3-triazolo 4,5-d!pyrimidine (13).

In order to dry it, 0.34 g (1.00 mmol) of 12 is evaporated twice in dry pyridine. The substance is dissolved in 4 ml of dry pyridine, and 0.41 g (1.20 mmol) of 4,4'-dimethoxytrityl chloride is added; the mixture is then stirred at 40° C. for 2 hours. After the mixture has been cooled down to room temperature, 5 ml of analytical grade methanol are added, and this mixture is stirred for a further 30 minutes. The reaction solution is concentrated down to approximately half its volume. 8 ml of a saturated solution of sodium hydrogen carbonate are then added, and the whole is extracted four times with 10 ml of methylene chloride on each occasion. The combined organic phases are extracted by shaking with 15 ml of a saturated solution of sodium chloride, dried over sodium sulfate and evaporated. The residue (0.73 g of a pale yellow foam) is further purified using column chromatography (column: 2×20 cm, silica gel, CH₂ Cl₂ /MeOH 95:5). Yield: 0.52 g (81%) of a colorless foam.

TLC (silica gel, CH₂ Cl₂ /MeOH=9:1): R_(f) =0.45. UV (methanol) λ_(max) (ε)=234 (29900), 275 (13800), 324 (24800). ¹ H-NMR (D₆ -DMSO) δ: 2.24 (s, N=CCH₃); 3.10 (m, H₂ -5'); 3.19 (s, N(CH₃)₂); 3.69 (s, (OCH₃)₂); 4.14 (m, H-4'); 4.51 (m, H-3'); 4.86 (m, H-2'); 5.28 (d, J=6.2 Hz, HO-3'); 5.68 (d, J=5.1 Hz, HO-2'); 6.25 (d, H-1'); 6.71-7.26 (m, 13 arom. H); 8.54 (s, H-5). C₃₄ H₃₇ N₇ O₆ calculated C 63.83 H 5.84 N 15.33. found C 63.64 H 5.84 N 15.31.

Example 18

7-{ 1-(Dimethylamino)ethylidene!amino}-3-{5-O-(4,4'-dimethoxytriphenylmethyl)-2-O- tris(1-methylethyl) silyl!-β-D-ribofuranosyl}-3H-1,2,3-triazolo 4,5-d!pyrimidine (14).

0.35 g (0.55 mmol) of the dried trityl compound 13 is initially introduced in 4 ml of dry pyridine. 140 mg (0.82 mmol) of silver nitrate are added to this solution, after which 145 μl (0.69 mmol) of triisopropyl chloride, which has previously been dissolved in 5 ml of tetrahydrofuran, are added under argon. The mixture is left to stir at room temperature with light being excluded. After 24 hours, a further 120 μl (0.55 mmol) of triisopropylsilyl chloride are added, and the mixture is left to stir at room temperature for a further 48 hours. The silver chloride which has precipitated out is filtered off and washed with a little tetrahydrofuran; 10 ml of a saturated solution of sodium hydrogen carbonate are then added to the filtrate. This is extracted four times with 10 ml of dichloromethane on each occasion, and the combined organic phases are dried over sodium sulfate. Following evaporation to dryness, 0.60 g is obtained of a faintly yellowish oil. The reaction products are purified and fractionated by means of column chromatography. (Column, 3×20 cm, silica gel, eluent ethyl acetate/petroleum ether 8:2). 0.31 g (71%) of a colorless foam are obtained from the more rapidly migrating main zone.

TLC (silica gel, ethyl acetate/petroleum ether 9:1): R_(f) =0.30. UV (methanol) λ_(max) (ε)=234 (29600), 274 (6800), 325 (25900). ¹ H-NMR (D₆ -DMSO) δ: 0.83-0.97 (m, Si- CH(CH₃)₂); 2.24 (s, N═CCH₃), 3.10 (m, H₂ -5'); 3.19 (s, N(CH₃)₂); 3.70 (s, (O--CH₃)₂); 4.19 (m, H-4'); 4.43 (m, H-3'); 5.21 (t, J=4.4 Hz, H-2'); 5.27 (d, J=6.3 Hz, HO-3'); 6.30 (d, J=4.3 Hz, H-1'); 6.75-7.35 (m, 13 arom. H); 8.53 (s, H-5). C₄₃ H₅₇ N₇ O₆ calculated C 64.87 H 7.23 N 12.23. found C 64.94 H 7.37 N 12.13.

Example 19

7-{ 1-(Dimethylamino)ethylidene!amino}-3-{5-O-(4,4'-dimethoxytriphenylmethyl)-3-O- tris(1-methylethyl)silyl!-β-D-ribofuranosyl}-3H-1,2,3-triazolo 4,5-d!pyrimidine (15).

0.08 g (18%) of a colorless foam are obtained from the more slowly migrating zone of the column chromatography which was carried out as described above for compound 14.

TLC (silica gel, ethyl acetate/petroleum ether 9:1): R_(f) =0.15. UV (methanol) λ_(max) (ε)=234 (29800), 274 (7400), 327 (24700). ¹ H-NMR (D₆ -DMSO) δ: 0.98 (s, Si CH(CH₃)₂ !₃); 2.19 (s, N═CCH₃); 3.10 (m, H₂ -5'); 3.18 (s, N(CH₃)₂); 3.68 (s, (OCH₃)₂); 4.17 (m, H-4'); 4.92 (m, H-3', H-2'); 5.64 (d, J=5.1 Hz, HO-2'); 6.27 (d, J=6.0 Hz, H-1'); 6.70-7.20 (m, 13 arom. H); 8.55 (s, H-5).

Example 20

7-{ 1-(Dimethylamino)ethylidene!amino}-3-{5-O-(4,4'-dimethoxytriphenylmethyl)-2-O- tris(1-methylethyl)silyl)-β-D-ribofuranosyl}-3H-1,2,3-triazolo 4,5-d!pyrimidine-3-O-phosphonate, triethylammonium salt (16).

0.67 g (9.75 mmol) of 1,2,4-triazole is added, under an argon atmosphere, to a solution of 114 μl (1.3 mmol) of phosphorus trichloride and 1.43 ml (13.0 mmol) of N-methylmorpholine in 10 ml of dry dichloromethane. After the reaction mixture has been stirred at room temperature for 30 min, it is cooled down to 0° C. and the silyl compound (14), dissolved in 2.5 ml of dry dichloromethane, is added dropwise within the space of 10 min. The reaction mixture is stirred at 0° C. for a further 20 min and then hydrolysed with 1M TBC buffer. The aqueous phase is extracted three times with 20 ml of dichloromethane on each occasion. The combined organic phases are dried over sodium sulfate and evaporated to dryness. The residue is chromatographed on a silica gel column (3×10 cm, CH₂ Cl₂ /MeOH/TEA 88:10:2). The fractions containing the product are together concentrated by evaporation, and the residue is taken up in 20 ml of dichloromethane; this solution is extracted by shaking four times with 5 ml of 0.1M TBC buffer on each occasion, dried over sodium sulfate and freed from the solvent. Yield: 0.21 g (83%) of a colorless foam.

TLC (silica gel, CH₂ Cl₂ /MeOH/TEA 88:10:2): R_(f) =0.6. UV (methanol) λ_(max) (ε)=234 (27300), 274 (11700), 325 (16400). ¹ H-NMR (D₆ -DMSO) δ: 0.75-0.95 (m, Si CH(CH₃)₂ !₃); 1.15; 2.99 (m, (CH₃ CH₂)₃ N); 2.24 (s, N═CCH₃); 3.20 (s, N(CH₃)₂); (H₂ -5' verdeckt); 3.69 (s, (O--CH₃)₂); 4.40 (m, H-4'); 4.79 (m, H-3'); 5.44 (m, H-2'); 5.50 u. 7.91 (d, ¹ J=602 Hz, P--H); 6.27 (d, J=6.0 Hz, H-1'); 6.76-7.40 (m, 13 arom. H); 8.50 (s, H-5); 10.90 (s, br, N⁺ --H). ³¹ P-NMR δ: 2.55 (dd, ¹ P_(PH) =602 H₂, ³ J_(PH) =9.5 H₂).

Example 21

Solid-phase synthesis of the oligoribonucleotides using the phosphonate method

The oligoribonucleotides are synthesized on a 1 mmol scale using the phosphonate technique and employing an Applied Biosystems, Weiterstadt, DNA synthesizer. Final oxidation is carried out manually.

1. The oligoribonucleotides are cleaved off from the CPG support, on the support column, by means of a 16-hour exposure to the action of ammonia (25% aqueous solution/ethanol 3:11).

2. Elimination of the base protective groups

The ammoniacal solution of the oligomers is heated in a water bath at 55° C. for 16 hours in the case of the unmodified (AU)₆ sequence, and at 40° C. for 3 hours in the case of the dodecamers containing 8-azaadenosine. The solutions are evaporated to dryness at room temperature, and the residues are coevaporated with abs. ethanol.

3. The 2'-silyl protective groups were eliminated by a 16-hour exposure, at room temperature, to the action of a one-molar solution of TBAF/THF.

Example 22 Synthesis of 5'-(z⁸ A-U)₆ -3' (17)

The 3'-phosphonate of 8-azaadenosine is employed for synthesizing the oligoribonucleotide 5'-(z⁸ A-U)₆ -3' (17). Here, compound (16) is employed together with 5'-(MeO)₂ Tr--, 2'-t-BuMe₂ Si-protected 3'-phosphonates of uridine. The oligonucleotides are synthesized on Controlled Pore Glass (CPG) in the 3' to 5' direction, with the 3'-terminally protected nucleoside being bound covalently to the solid phase via a succinyl spacer. At the beginning of the synthesis, the 5'Dmt group of the support-bound nucleoside is eliminated using 2.5% dichloroacetic acid in dichloromethane. Coupling then takes place with the phosphonate which has been activated with pivaloyl chloride. In order to avoid incorrect sequences, unreacted 5'-OH groups are reacted with isopropyl phosphite.

Example 23

Purification of the oligoribonucleotides

1.) Preliminary desalting

Using Qiagen tip 500 anion exchange columns. A ®Qiagen column is equilibrated with 5 ml of 0.1M TBC buffer, loaded with the oligomer solution and washed with 5 ml of 0.1M TBC buffer. After that, the oligomers are eluted from the column with 1M TBC buffer. The product fractions are detected using UV/TLC plates. The buffer solutions are removed from the oligomer-containing fractions using a ®Speed Vac centrifuge under vacuum.

2.) Preparative HPLC

The oligomers were isolated by reverse phase HPLC on a RP 18 ®LiChrosorb column. For this purpose, the oligomer is taken up in 400 μl of a 1% aqueous solution of diethyl pyrocarbonate (DEPC), and this sample is then heated at 95° C. for 2-3 min, and then cooled rapidly to 0° C., in order to prevent the formation of secondary structures. DEPC is an RNase inhibitor. A sample (10 μl) of this solution is then injected in order to determine the retention times.

The solution is then added to the RP-18 column in portions of 50-100 μl; the main peak is then separated off, and the combined fractions are concentrated down to a volume of approximately 5 ml.

Mobile phases:

A: 0.1M TEAA (sterile, pH 7.5)/CH₃ CN 95:5

B: CH₃ CN

System I: 20 min, 0-20% B in A

System II: 30 min, 0-20% B in A

Retention time of oligomer 17

    ______________________________________                                         Oligomer     Retention time  min!                                                                         System                                              ______________________________________                                         (z.sup.8 A-U).sub.6                                                                         28.6          II                                                  ______________________________________                                    

Flow rate: 1 ml/min

3. Desalting

The 5 ml of oligomer solution are added to the column (Millipore, Eschborn) which has previously been autoclaved and equilibrated with 5 ml of CH₃ CN, 5 ml of 0.05M TEAA buffer (pH 7.0)/CH₃ CN 1:1, and with 5 ml of 0.05M TEAA buffer; the column is then washed with 5 ml of 0.05M TEAA solution, and the oligoribonucleotides are eluted from the column using a mixture of MeOH/CH₃ CN/H₂ O 1:1:1 in portions of 1 ml. The oligomer-containing fractions are identified by HPLC. After having been lyophilized on a ®Speed Vac concentrator, the oligoribonucleotides are stored at -25° C.

Example 24

Total hydrolysis of the oligoribonucleotides

0.2 A₂₆₀ units of the oligomers are dissolved in 200 μl of tris-HCl buffer (pH 8.3), after which 4 μg (2 μl) of snake venom phosphodiesterase (Boehringer Mannheim) are added and the mixture is incubated at 37° C. for 30 min. Following the addition of 3 μg (5 μl) of alkaline phosphatase, the solution is maintained at 37° C. for a further 15 min. The nucleoside composition of the reaction solution is then determined by means of HPLC (RP-18 column; mobile phase: 0.1M TEAA buffer/CH₃ CN 95:5, flow rate 1 ml/min).

Retention times of the nucleosides:

A=11.4 min z⁸ A=10.0 min l=4.8 min U=3.6 min

The HPLC peak areas are divided by the respective extinction coefficients and then related to each other.

Extinction coefficients at 260 nm:

ε (A)=15300, ε (z8A)=7100, ε (U)=10200, ε (l)=7400.

Example 25

Fractosil®-bound N⁸ -8-Aza-2'-deoxyguanosine.

p-Nitrophenol (7 mg, 0.05 mmol) and N,N-dicyclohexylcarbodiimide (10 mg, 0.048 mmol) were added to a solution of the 3'-O-succinate (6) from Example 6 (30 mg, 0.04 mmol) in 1,4-dioxane/5% pyridine (1 ml). After the mixture had been stirred at room temperature for 2 hours, the filtrate from the solution was added to a suspension of Fractosil 200® (80 mg, 450 μmol/g; Merck) in DMF (1 ml). After triethylamine (100 μl) had been added, the mixture was shaken for 4 hours with acetic anhydride (20 μl) being added during this period. The polymeric support was filtered off, washed with 30 ml each of DMF, ethanol and ether, and dried in vacuo. In order to determine the yield of the polymer-bound nucleoside, the substance was taken up with 0.1M p-toluenesulfonic acid (5 ml) in MeCN. The loading of the support was calculated, by UV spectrophotometry from the extinction at 498 nm (Dmt=70000), to be 64 μmol of 8-aza-2'-deoxyguanosine/g of Fractosil®.

Example 26

Solid-phase synthesis of the oligodeoxyribonucleotides using the phosphonate method.

The oligodeoxyribonucleotide syntheses were carried out, on solid phase (CPG: ®Controlled Pore Glass) and on a 1 μmol scale, using the phosphonate technique and employing a 380 B DNA synthesizer (Applied Biosystems, Weiterstadt), with the DNA fragment being synthesized in the 3'-5' direction. Here, the oxidation cycle (detritylation, coupling, capping and oxidation) followed a program which was developed for phosphonate chemistry H. Koster, K. Kulikowsky, T. Liese, W. Heikens, V. Kohli, Tetrahedron 1981, 37, 363!. The base-protected oligonucleotide, which was also Dmt-protected on the 5'-hydroxyl group, was cleaved off from the support within 30 min using 25% aqueous ammonia. Following a further addition of aqueous ammonia (I ml, 25%), the protective groups on the heterocycles were eliminated within 24 hours at 60° C. After having added a drop of triethylamine (prevention of the premature elimination of the 5'-OH protective group), the samples were concentrated to about 200 μl in a Speed Vac ®Concentrator. In this state, they keep for some months at -25° C.

Example 27

Solid-phase synthesis of the oligodeoxyribonucleotides using the phosphoramidite method.

The oligodeoxyribonucleotide syntheses were carried out, on a 1 μmol scale, by means of the solid-phase phosphoramidite technique, on an automated 380 B DNA synthesizer (Applied Biosystems, Weiterstadt) using ®CPG (Controlled Pore Glass) or ®Fractosil to which the first nucleoside unit was bound by its 3' end. In this case, the following steps were carried out:

1. washing with abs. acetonitrile,

2. treating with 3% trichloroacetic acid in dichloromethane,

3. washing with abs. acetonitrile,

4. condensing with 10 μmol of 5'-O-dimethoxytritylnucleoside-3'-β-cyanoethyl phosphite-diisopropyl amidite and 50 μmol of tetrazole in 0.3 ml of abs. acetonitrile,

5. washing with acetonitrile,

6. capping with 20% acetic anhydride in THF containing 40% lutidine and 10% dimethylaminopyridine,

7. washing with acetonitrile,

8. oxidizing with iodine (1.3 g in THF/water/pyridine; 70:20:5 =v:v:v).

Steps 1 to 8, termed a DNA reaction cycle below, were repeated in order to construct the oligonucleotide which corresponded to the sequence to be synthesized, with the 5'-O-dimethoxytrityl(nucleoside base)-3'-β-cyanoethyl phosphite-diisopropylamidite corresponding to the sequence being in each case employed in step 4. Once the synthesis is complete, working-up takes place as described in Example 8.

Example 28

Synthesis of d(Cz⁸ GCGCG).

The synthesis was carried out as described in Example 25, proceeding from CPG-bound 5'-O-dimethoxytrityl-2'-deoxyguanosine. The first three nucleotide addition steps are carried out using commercially available 5'-O-dimethoxytrityl(nucleoside base)-3'-H-phosphonates. In order to introduce the 8-aza-2'-deoxyguanosine, 3'- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{(dimethylamino)methylidene!amin}-3H-1,2,3-triazolo 4,5d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 was used in the fourth condensation cycle.

Example 29

Synthesis of d(Cz⁸ GCz⁸ GCG).

The synthesis was carried out in a manner analogous to that described in Example 28, with 3- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 being in each case employed in the second and fourth condensation cycle in order to introduce the 8-aza-2'-deoxyguanosine.

Example 30

Synthesis of d(GCz⁸ GCGC).

The synthesis was carried out in a manner analogous to that described in Example 29, proceeding from a CPG support which was loaded with cytidine, with 3- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 being employed in the third condensation cycle in order to introduce the 8-aza-2'-deoxyguanosine.

Example 31

Synthesis of d(Tz⁸ GGGGT).

The synthesis was carried out in an analogous manner to that described in Example 28, proceeding from CPG-bound 5'-O-dimethoxytritylthymidine, with3- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 being used in the fourth condensation cycle to introduce the 8-aza-2'-deoxyguanosine.

Example 32

Synthesis of d(TGz⁸ GGGT).

The synthesis was carried out in an analogous manner to that described in Example 30, proceeding from CPG-bound 5'-O-dimethoxytritylthymidine, with 3- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 being used in the third condensation cycle in order to introduce the 8-aza-2'-deoxyguanosine.

Example 33

Synthesis of d(Tz⁸ Gz⁸ Gz⁸ Gz⁸ GT)

The synthesis was carried out in an analogous manner to that described in Example 30, proceeding from CPG-bound 5'-O-dimethoxytritylthymidine, with3- 2-deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(triethylammonium phosphonate) (4) from Example 4 being in each case used in condensation cycles 1 to 4 in order to introduce the 8-aza-2'-deoxyguanosine.

Example 34

Synthesis of d(GTAz⁸ GAATTCTAG).

The synthesis was carried out as described in Example 26, proceeding from CPG-bound 5'-O-dimethoxytrityl-2'-deoxyguanosine. 3- 2-Deoxy-5-O-(4,4'-dimethoxytrityl)-β-D-erythropentofuranosyl!5-{ (dimethylamino)methylidene!amino}-3H-1,2,3-triazolo 4,5-d!pyrimidin-7-(6H)-one 3'-(trimethylammonium phosphonate) (4) from Example 4 was used in the eighth condensation cycle in order to introduce the 8-aza-2'-deoxyguanosine.

Example 35

Purification of the trityl-protected and deprotected oligonucleotides using HPLC.

The Dmt-protected oligomers were purified by HPLC on RP-18 silica gel (eluent system I), in a first purification step, and evaporated to dryness in vacuo at 40° C. A subsequent, 20-minute treatment with 250 μl of 80% acetic acid resulted in the elimination of the 5'-trityl group. In a second purification step, the oligomers, which were now completely deprotected, were purified for a second time by RP-18 HPLC (eluent system II). The combined main zones were evaporated and the residue was dissolved in approximately 500 μl of water; this solution was then desalted through a short RP-18 column (eluent system III). Following lyophilization, the oligomers (5-20 A₂₆₀ units) were taken up in 100 μl of water, and these solutions were stored at -25° C. Use was made of the following eluent systems composed of:

0.1M triethylammonium acetate, pH 7.0/5% acetonitrile (A)

acetonitrile (B)

water (C)

methanol/water (3:2) (D)

I: 20 min (0-20% B) in A

II: 20 min (15-40% B) in A

III: 15 min C, 10 min D

IV: 100% A

V: 100% B

The oligomers were observed to have the following retention times:

    ______________________________________                                                                 Retention time                                         Oligomer       Example   min!       Eluent                                     ______________________________________                                         d(Cz.sup.8 GCGCG)                                                                             10       15.1 (12.5) I (II)                                     d(Cz.sup.8 GCz.sup.8 GCG)                                                                     11       15.8 (12.9) I (II)                                     d(GCz.sup.8 GCGC)                                                                             12       15.5 (12.5) I (II)                                     d(Tz.sup.8 GGGGT)                                                                             13       13.4 (12.2) I (II)                                     d(TGz.sup.8 GGGT)                                                                             14       13.5 (12.1) I (II)                                     d(Tz.sup.8 Gz.sup.8 Gz.sup.8 GZ.sup.8 GT)                                                     15       13.6 (12.4) I (II)                                     d(GTAz.sup.8 GAATTCTAG)*                                                                      16       15.0 (12.4) I (II)                                     ______________________________________                                          *= (Seq. ID. No. 30)                                                     

Example 36

Characterization of the oligodeoxyribonucleotides by means of enzymic hydrolysis.

0.2 A₂₆₀ units of the oligomers were dissolved in 0.1M tris/HCl buffer (pH 8.3, 200 μl) and incubated with snake venom phosphodiesterase (EC 3.1.4.1, Crotallus durissus, Boehringer Mannheim; 6 μg) at 37° C. for 45 min, and with alkaline phosphatase (EC 3.1.3.1, calf liver, Boehringer Mannheim; 2 μg) at 37° C. for 30 min. The hydrolysis products were detected at 260 nm using reverse-phase HPLC (RP-18, eluent IV). The composition of the oligodeoxyribonucleotides was quantified using the peak areas and the extinction coefficients of the nucleosides (ε₂₆₀ : dA 15400, dC 7300, dG 11700, dT 8800, z⁸ G_(d) 12000).

Example 37 Determination of the enzymic hypochromicity.

The UV absorption at 260 nm of approximately 0.2 A₂₆₀ units of the oligomers was determined in 0.1M tris/HCl buffer (pH 8.3, 200 μl) before and after adding snake venom phosphodiesterase (10 μg). Taking into consideration the absorption of the enzyme, the hypochromicity is given by the relationship:

    H.sub.enzym. =(.sub.monomer -.sub.oligomer)(.sub.monomer).sup.-1 !×100

Example 38

UV-spectroscopic and CD-spectroscopic determinations of the T_(m) values, and calculation of the thermodynamic data.

The T_(m) values of the oligomers were determined using a Cary 1 UV/vis spectrophotometer (Varian, Melbourne, Australia). The temperature was varied linearly by 0.5° C. or 1.0° C. per minute. For investigating the melting temperature, oligomer concentrations of between 0.2-0.8 A₂₆₀ units in 1 ml of 60 mM of sodium cacodylate buffer (pH 7.5, 1M NaCl, 100 mM MgCl₂) were used. In the experiments carried out on the nonself-complementary oligonucleotides, the single-strand concentration was 0.2-0.6 OD. The melting hypochromicity in % is obtained from the change in absorption before and after melting, in accordance with the following equation:

    H.sub.melt = (A.sub.e -A.sub.t)A.sub.e.sup.-1 !×100

The melting curves were analysed using a program based on a two-state model (stacked/unstacked) in accordance with the equation:

    lnK=ln (E.sup.S -E)/(E.sup.U -E)!=S/R-H/RT

where E=absorption at the relevant wavelength, S=stacked and U=unstacked. The temperature-dependent CD spectra were plotted on a Jasco 600 spectropolarimeter, using a temperature-regulatable quartz cuvette, in a wavelength range of 200-350 nm. The temperature was increased in intervals of 5°-10° C. in a range of 5°-80° C., in concentrations of 3-15 μM in 60 mM Na cacodylate buffer, and also with 0.1M, 1M and 4M NaCl.

Example 39

T_(m) values and hypochromicity data for duplex formation a)

    ______________________________________                                         Oligomer       Tm  °C.!                                                                         Hypochromicity  %!                                     ______________________________________                                         d(CGCGCG)      45       22                                                     d(GCGCGC)      45       29                                                     d(Cz.sup.8 GCGCG)                                                                             49       23                                                     d(Cz.sup.8 GCz.sup.8 GCG)                                                                     52       21                                                     d(GCz.sup.8 GCGC)                                                                             47       27                                                     ______________________________________                                          a) Measured in 1M NaCl, 100 mM MgCl.sub.2, 60 mM cacodylate buffer, pH         7.0.                                                                     

Example 40

Testing for nuclease stability

10 nmol of the oligonucleotide to be investigated are dissolved in 450 μl of 20% fetal calf serum in RPMI medium and 50 ml of doubly distilled water, and this solution is incubated at 37° C. 10 μl samples for gel electrophoresis and 20 μl samples for HPLC are then removed immediately and after 1, 2, 4, 7 and 24 hours, with these samples then being treated with 5 μl or 10 μl, respectively, of formamide to terminate the reaction and then being heated at 95° C. for 5 minutes. For the gel electrophoresis, the samples are loaded onto a 15% polyacrylamide gel (2% BIS), which is then developed at approximately 3000 volt hours. The bands are visualized by silver staining. For the HPLC analysis, the samples are injected into a Gene-Pack Fax HPLC column (Waters/Millipore) and chromatographed at 1 ml/min using from 5 to 50% buffer A in B (buffer A: 10 mM sodium dihydrogen phosphate, 0.1M NaCl in acetonitrile/water 1:4 (v:v), pH 6.8; buffer B: as A, but containing 1.5M NaCl.

Example 41 Testing for antiviral activity:

The antiviral activity exerted by the test substances against various herpes viruses which are pathogenic to humans is investigated in a cell culture test system. For the experiment, monkey kidney cells (Vero, 2×10⁵ /ml) in serum-containing Dulbecco's MEM (5% fetal calf serum, FCS) are sown in 96-well microtiter plates, and incubated at 37° C. and 5% CO₂ for 24 h. The serum-containing medium is then sucked off and the cells are flooded twice with serum-free Dulbecco's MEM (-FCS). Prior to use, the test substances are diluted in H₂ 0 to a concentration of 600 μM and stored at -18° C. For the test, further dilution steps are carried out in Dulbecco's minimal essential medium (MEM). In each case, 100 μl of the individual test substance dilutions are added to the rinsed cells together with 100 μl of serum-free Dulbecco's MEM (-FCS). After having been incubated at 37° C. and 5% CO₂ for 3 h, the cells are infected with herpes simplex virus type 1 (ATCC VR733, HSV-1 F strain) or with herpes simplex virus type 2 (ATCC VR734, HSV-2 G strain) in concentrations at which the cell lawn is completely destroyed within the space of 3 days. In the case of HSV-1, the magnitude of the infection is 500 plaque-forming units (PFU) per well, and in the case of HSV-2 350 PFU/well. The experimental samples then contain test substance in concentrations of from 80 μM to 0.04 μM in MEM, supplemented with 100 U/ml penicillin G and 100 mg/l streptomycin. All experiments are carried out in duplicate with the exception of the controls, eight of which are carried out per plate. The experimental samples are incubated at 37° C. and 5% CO₂ for 17 h. The cytotoxicity of the test substances is determined, after a total incubation time of 20 h, by microscopic assessment of the cell cultures. The highest preparation concentration which fails to elicit any microscopically recognizeable cell damage under the given experimental conditions is designated the maximum tolerated dose (MTD). After that, FCS is added to a final concentration of 4%, and the plates are incubated for a further 55 h at 37° C. and 5% CO₂. The untreated infection controls then exhibit a complete cytopathic effect (CPE). Once the cell cultures have been assessed microscopically, they are then stained with neutral red using the vital staining method of Finter (1966). The antiviral activity of a test substance is defined as the minimum inhibitory concentration (MIC) which is required in order to protect 30-60% of the cells from the cytopathic effect due to the virus.

Abbreviations:

    ______________________________________                                         A          adenosine                                                           bz         benzoyl                                                             br.        broad                                                               calc.      calculated                                                          CD         circular dichroism                                                  d          doublet                                                             dG         2'-deoxyguanosine                                                   dA         2'-deoxyadenosine                                                   dC         2'-deoxycytidine                                                    dT         2'-deoxythymidine                                                   DEPC       diethyl pyrocarbonate                                               DMA        dimethylacetamide                                                   (D.sub.6)DMSO                                                                             dimethyl sulfoxide, deuterated 6 times                              DMF        dimethylformamide                                                   DNA        deoxyribonucleic acid                                               Dmt        4,4'-dimethoxytrityl, (4,4'-dimethoxytriphenyl-                                methyl)                                                             EDTA       ethylenediamine tetraacetate                                        EtOAc      ethyl acetate                                                       Et.sub.3 N triethylamine                                                       FC         flash chromatography                                                G          free enthalpy                                                       h          hour                                                                H          duplex formation enthalpy                                           HPLC       high pressure liquid chromatography                                 Hyp.       hypochromicity                                                      I          inosine                                                             ibu        isobutyryl                                                          J          coupling constant                                                   K.sub.m    Michaelis-Menten constant                                           M.p.       melting point                                                       NMR        nuclear magnetic resonance                                          PAGE       polyacrylamide gel electrophoresis                                  PCR        polymerase chain reaction                                           ppm        parts per million                                                   2-P-OH     isopropanol                                                         R.sub.f    retention in TLC relative to the eluent front                       RNA        ribonucleic acid                                                    RP         reverse phase                                                       R.T.       room temperature                                                    s          singlet                                                             S          duplex formation entropy                                            SVPD       snake venom phosphodiesterase                                       t          triplet                                                             TBAF       tetrabutylammonium fluoride                                         TBC        triethylammonium bicarbonate                                        TEP        triethylammonium phosphonate                                        TLC        thin layer chromatography                                           Tms        tris(1-methylethyl)silyl                                            Tris       tris(hydroxymethyl)aminomethane                                     T.sub.m    oligomer melting temperature                                        U          uridine                                                             UV         ultraviolet                                                         V.sub.max  maximum reaction velocity                                           z.sup.8 A  8-azaadenosine                                                      z.sup.8 G  8-aza-2'-deoxyguanosine                                             λ   wavelength                                                          ε  molar extinction coefficient                                        ______________________________________                                    

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 30                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       ACACCCAATTCTGAAAATGG20                                                         (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       AGGTCCCTGTTCGGGCGCCA20                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..28                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       GTCGACACCCAATTCTGAAAATGGATAA28                                                 (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..25                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       GCTATGTCGACACCCAATTCTGAAA25                                                    (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..31                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       TCGTCGCTGTCTCCGCTTCTTCTTCCTGCCA31                                              (2) INFORMATION FOR SEQ ID NO: 6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HIV                                                              (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..31                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       CTGTCTCCGCTTCTTCTTCCTGCCATAGGAG31                                              (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HSV-1                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       GCGGGGCTCCATGGGGGTCG20                                                         (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..15                                                            (D) OTHER INFORMATION: /note= "c-Ha-ras"                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       CAGCTGCAACCCAGC15                                                              (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..21                                                            (D) OTHER INFORMATION: /note= "c-myc"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       GGCTGCTGGAGCGGGGCACAC21                                                        (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..15                                                            (D) OTHER INFORMATION: /note= "c-myc"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      AACGTTGAGGGGCAT15                                                              (2) INFORMATION FOR SEQ ID NO: 11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..15                                                            (D) OTHER INFORMATION: /note= "c-myb"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      CACGTTGAGGGGCAT15                                                              (2) INFORMATION FOR SEQ ID NO: 12:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "c-myb"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                      GTGCCGGGGTCTTCGGGC18                                                           (2) INFORMATION FOR SEQ ID NO: 13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mous                                                             (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "c-myb"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      GTGTCGGGGTCTCCGGGC18                                                           (2) INFORMATION FOR SEQ ID NO: 14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..21                                                            (D) OTHER INFORMATION: /note= "c-fos"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      GGAGAACATCATGGTCGAAAG21                                                        (2) INFORMATION FOR SEQ ID NO: 15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..22                                                            (D) OTHER INFORMATION: /note= "c-fos"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      CCCGAGAACATCATGGTCGAAG22                                                       (2) INFORMATION FOR SEQ ID NO: 16:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /note= "c-fos"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      GGGGAAAGCCCGGCAAGGGG20                                                         (2) INFORMATION FOR SEQ ID NO: 17:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /note= "p-120"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                      CACCCGCCTTGGCCTCCCAC20                                                         (2) INFORMATION FOR SEQ ID NO: 18:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "EGF-Rezeptor"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                      GGGACTCCGGCGCAGCGC18                                                           (2) INFORMATION FOR SEQ ID NO: 19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /note= "EGF-Rezeptor"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                      GGCAAACTTTCTTTTCCTCC20                                                         (2) INFORMATION FOR SEQ ID NO: 20:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "p53 Tumorsuppressor"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                      GGGAAGGAGGAGGATGAGG19                                                          (2) INFORMATION FOR SEQ ID NO: 21:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..21                                                            (D) OTHER INFORMATION: /note= "p53 Tumorsuppressor"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                      GGCAGTCATCCAGCTTCGGAG21                                                        (2) INFORMATION FOR SEQ ID NO: 22:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "cdc2-Kinase"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      GTCTTCCATAGTTACTCA18                                                           (2) INFORMATION FOR SEQ ID NO: 23:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "PCNA (proliferating cell                        nuclear antigen)"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                      GATCAGGCGTGCCTCAAA18                                                           (2) INFORMATION FOR SEQ ID NO: 24:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..18                                                            (D) OTHER INFORMATION: /note= "VLA-4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                      GCAGTAAGCATCCATATC18                                                           (2) INFORMATION FOR SEQ ID NO: 25:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /note= "ICAM"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                      CCCCCACCACTTCCCCTCTC20                                                         (2) INFORMATION FOR SEQ ID NO: 26:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..20                                                            (D) OTHER INFORMATION: /note= "ICAM"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                      CTCCCCCACCACTTCCCCTC20                                                         (2) INFORMATION FOR SEQ ID NO: 27:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..19                                                            (D) OTHER INFORMATION: /note= "ICAM"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                      GCTGGGAGCCATAGCGAGG19                                                          (2) INFORMATION FOR SEQ ID NO: 28:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..21                                                            (D) OTHER INFORMATION: /note= "ELAM-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                      ACTGCTGCCTCTTGTCTCAGG21                                                        (2) INFORMATION FOR SEQ ID NO: 29:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: exon                                                             (B) LOCATION: 1..22                                                            (D) OTHER INFORMATION: /note= "ELAM-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                      CAATCAATGACTTCAAGAGTTC22                                                       (2) INFORMATION FOR SEQ ID NO: 30:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (iii) HYPOTHETICAL: YES                                                        (iii) ANTI-SENSE: YES                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                                (B) LOCATION: 1..11                                                            (D) OTHER INFORMATION: /note= "N = 8-Azaguanin"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                      GTANAATTCTAG12                                                                 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We claim:
 1. A nucleotide monomer of the formula V ##STR19## in which V is oxy, sulfanediyl or imino;Y^(b) is oxy, sulfanediyl, or methylene; a is oxy or methylene; R^(2b) is hydrogen, OR¹², C₁ -C₁₈ -alkoxy, C₁ -C₆ -alkenyloxy, halogen, azido or NR¹⁰ R¹¹ ; R¹ is a protective group which is customary in nucleotide chemistry; R^(1b) is a radical of the formula IIc or IId ##STR20## in which U is O--R⁷ or S--R⁷ ; Q is a radical --NR⁸ R⁹ ; R⁷ is --(CH₂)₂ --CN; R⁸ and R⁹ are identical or different and are C₁ -C₆ -alkyl, or, together with the nitrogen atom carrying them, are a 5-9-membered heterocyclic ring which can additionally contain a further heteroatom selected from the group O, S, and N, E' and F' are, independently of each other, H, OH, or NR¹⁰ R¹¹, R¹⁰ and R¹¹ are identical or different and are hydrogen or an amino protective group which is customary in nucleotide chemistry, or R¹⁰ and R¹¹ together form an amino protective group which is customary in nucleotide chemistry, R¹² is a hydroxyl protective group which is customary in nucleotide chemistry, and the curved bracket indicates that R^(2b) and the adjacent phosphoryl radical can be in the 2' and 3' positions, or else, conversely, in the 3' and 2' positions.
 2. A process for preparing a nucleotide monomer of the formula V as claimed in claim 1, said process comprising the steps of:introducing onto an unprotected nucleotide monomer of the formula V suitable amino protective groups or hydroxyl protective groups, and converting the resulting protected monomers into the corresponding phosphonate derivatives or phosphoamidite derivatives thereof.
 3. A method for preparing oligonucleotides which form stable hybrids with their target nucleic acids, said method comprising condensing nucleotide monomers of formula V according to claim 1 onto an appropriately derivatized support or onto a growing oligomer chain.
 4. A compound of the formula VI ##STR21## in which, independently of each other, U'=U"=U"' is hydroxyl or mercapto;e and f are 0 or 1; R¹³ is hydrogen or OH, and E and F are H, OH or NH₂, excluding compounds of the formula VI in which U', U", U"', R¹³ and F are OH, E is NH₂, and e and f are
 1. 5. A nucleotide monomer of formula V according to claim 1, whereinR^(2b) is hydrogen, OR¹², C₁ -C₁₈ -alkoxy, allyloxy, halogen, azido or NR¹⁰ R¹¹, R⁸ and R⁹ are isopropyl or ethyl, R¹² is t-butyldimethylsilyl, triisopropylsilyl, o-nitrobenzyl, p-nitrobenzyl, tris(1-methylethyl)silyl or 2-fluorophenyl-4-methoxy-piperidin-4-yl.
 6. A nucleotide monomer of formula V according to claim 1, wherein when R⁸ and R⁹ are a 5-9-membered heterocyclic ring, the ring additionally contains a further heteroatom selected from ##STR22## 